"
Team:Mexico-UNAM-IPN/Horizontal-Transfer/30 September 2008
From 2008.igem.org
(Difference between revisions)
m (incomplete, pause for lunch) |
(1st complete entry) |
||
| Line 2: | Line 2: | ||
#Dilution 1:100 in 30ml of sterile LB media. Incubate for 2.5-3hrs at 37°C. | #Dilution 1:100 in 30ml of sterile LB media. Incubate for 2.5-3hrs at 37°C. | ||
#Thaw on ice for for 10 minutes. transfer to 12 vials. | #Thaw on ice for for 10 minutes. transfer to 12 vials. | ||
| - | #Centrifugate for 7min at 4°C at 3500rpm. Eliminate supernatant and decant. fill the vials. | + | #Centrifugate for 7min at 4°C at 3500rpm. Eliminate supernatant and decant. fill the vials with the culture and centrifugate again. Repeat ≈ 3 times. |
| + | #Resuspend the pellet in 1ml of sterile cold 10mM MgCl<small>2</small>in each vial. Add 500µl of 10mM MgCl<small>2</small> to eeach vial. | ||
| + | #Centrifugate at 3500 rpm for 7min at 40°C. | ||
| + | #Eliminate the supernatant and resuspend in 100µl of 50mM CaCl<small>2</small>, Glycerol at 20%. Add 100µl of 50mM CaCl<small>2</small>, Glycerol at 20%. Incubate 1hr at 4°C. | ||
| + | #Transfer to 0.5ml eppendorf tubes and freeze. | ||
Revision as of 21:50, 27 October 2008
- Culture o/n of JM101 and JM109 strains.
- Dilution 1:100 in 30ml of sterile LB media. Incubate for 2.5-3hrs at 37°C.
- Thaw on ice for for 10 minutes. transfer to 12 vials.
- Centrifugate for 7min at 4°C at 3500rpm. Eliminate supernatant and decant. fill the vials with the culture and centrifugate again. Repeat ≈ 3 times.
- Resuspend the pellet in 1ml of sterile cold 10mM MgCl2in each vial. Add 500µl of 10mM MgCl2 to eeach vial.
- Centrifugate at 3500 rpm for 7min at 40°C.
- Eliminate the supernatant and resuspend in 100µl of 50mM CaCl2, Glycerol at 20%. Add 100µl of 50mM CaCl2, Glycerol at 20%. Incubate 1hr at 4°C.
- Transfer to 0.5ml eppendorf tubes and freeze.
